1. Field of the Invention
The present invention relates to a method for the quantitative determination of a monosaccharide, such as sialic acid, constituting a sugar composition.
2. Description of Related Art
Sugar compositions such as glycoproteins and glycolipids are important substances that constitute a mechanism of function-regulation for human body, particularly immunity related portions and, recently, investigations have been being made intensively in the top front of medicine, such as therapy of AIDS or tumors. It has already been considered that the sugar chain of glycoprotein present on the surface of AIDS virus bonds to the CD4 molecule on the surface of host cells to cause infection and there have been many reports that the sugar moiety of the sugar composition varies due to tumors.
Among such methods, particularly quantitative determination of various monosaccharides constituting the sugar composition composed mainly of sialic acid is the key of the investigation and various methods have been proposed for achieving more rapid and higher sensitivity analysis.
Various conventional methods and their advantages and disadvantages will be explained below.
For example,
(1) A method for determining sialic acid, neutral sugars and amino sugars by gas-liquid chromatography (GLC) after converting the sample into a volatile sugar derivative by methanolysis and trimethylsilylation: PA0 (2) A method for the determination of sialic acid in which sialic acid liberated by sialidase or an acid is converted to 1,2-diamino-4,5-methylenedioxybenzene acid (DMB) derivative, followed by high performance liquid chromatography (HPLC): PA0 (3) A method for the determination of amino sugars by amino acid analysis: PA0 (4) A method for the determination of neutral sugars and amino sugars that involves labeling the sugars with pyridylamino, converting them into ethyl 4-aminobenzoate derivatives, followed by HPLC: PA0 (5) A method for the determination of sialic acid, neutral sugars and amino sugars by converting the sialic acid and sugars into 1-phenyl-3-methyl-5-pyrazolone (PMP) derivative, followed by HPLC: PA0 (6) A method for the determination of sialic acid, neutral sugars and amino sugars that involves converting the sialic acid and sugars into cyanoacetamide derivatives after isolation by HPLC: PA0 (7) A method for the determination of sialic acid, neutral sugars and amino sugars using anion exchange chromatography and an electrochemical detector:
This method has defects that although it can determine sialic acid, neutral sugars and amino sugars simultaneously, it requires a large amount of sample (10 nmol or more as monosaccharide), that the analysis by GLC is not suitable for treating multiple samples, and that N-acetylglucosamine bonded to an asparagine residue cannot be determined (Anal. Biochem., 119, 17-24 (1982)).
This method is simple and has high sensitivity. However, it can analyze only sialic acid and the derivative is unstable and has to be analyzed within 12 hours ("Method for Studying Glycoprotein Sugar Chain (Biochemistry Experimental Methods 23)", Gakkai Shuppan Center, p.20 (1989)) and is limited in its application.
This method is simple and has high sensitivity but it can analyze only amino sugars.
This method cannot analyze sialic acid.
This method can be determined with high sensitivity, but has such an inconvenience that sialic acid can not be simultaneously determined together with neutral sugars and amino sugars.
This method is a post column labeling method and is simple but takes a long time for analysis and cannot afford simultaneous determination of sialic acid together with neutral sugars and amino sugars so that it is inconvenient.
This method can save the trouble of labeling but it is inconvenient in that it requires a special apparatus and that it cannot determine sialic acid together with neutral sugars and amino sugars simultaneously (Methods in Enzymology, 230, 208-225 (1994)).
As stated above, conventional methods for the determination of monosaccharides constituting sugar compositions are advantageous in a way and disadvantageous in another. Whereas the method for the determination of sugar compositions that enables diagnosis of affected sites without surgical invasion gives less stress to patients and hence are being increasingly used. Thus, a method that can treat many samples in a simple manner is desired. Accordingly, there has been a keen desire for urgently developing a method for simultaneous determination of monosaccharides that constitute sugar compositions including sialic acid that has recently attracted much attention as an indices for monitoring changes in pathological state affected by, e.g., tumors, with high rate, high sensitivity and accuracy.